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Inducible interleukin-12

a technology of interleukin-12 and interleukin, which is applied in the field of inducible interleukin-12, can solve the problems of severe toxicity of most of the enrolled patients, only limited clinical trials results of recombinant il-12, and death of two patients, and achieve the effect of treating or preventing cancer

Active Publication Date: 2013-10-15
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although recombinant IL-12 has demonstrated potent anti-cancer activity in animal models, recombinant IL-12 has provided only limited results in clinical trials (Del Vecchio et al.
In addition, systemic administration of IL-12 in a phase II clinical trial for renal cell carcinoma resulted in severe toxicity for most of the enrolled patients and the deaths of two patients (Cohen, J.

Method used

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  • Inducible interleukin-12
  • Inducible interleukin-12
  • Inducible interleukin-12

Examples

Experimental program
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Effect test

example 1

[0074]Human single chain IL-12 (hscIL-12) is synthesized by the company GeneArt (codon optimized, SEQ ID NO: 1) and Epoch® Biolabs Inc. (wildtype, SEQ ID NO: 2) by linking the p40 subunit sequence and p35 subunit sequence with an amino acid linker of six Glycines and one Serine. The hscIL-12 is cloned into MSGV1 retrovirus vector using the Nco I and Xho I restriction sites to generate MSGV1-hIL12. The NFAT promoter, containing six repeating NFAT-binding motifs and a minimal IL-2 promoter (SEQ ID NO: 4), is cut from the pSIN-(NFAT)6-GFP vector (Erik, H. et al., Blood, 96(2): 459-66(2000)) by Xho I and Bam HI and used to replace the PGK promoter of the pRRLSIN.cPPT.PGK.GFP lentivirus vector (Gregory L. et al., Human Gene Ther., 14:497-507(2003)) digested with the same enzyme to generate a pRRLSIN.cPPT.NFAT.GFP vector.

[0075]The vector pRRLSIN.cPPT.NFAT.hscIL12 (SEQ ID NO: 5) is constructed by ligating three fragments: the NFAT promoter from the pRRLSIN.cPPT.NFAT.GFP vector cut with Xho...

example 2a

[0078]The pSERS11.NFAT.hIL12-F vector (SEQ ID NO: 6) is constructed by excising the GFP expression cassette from the pSERS11MP.GFP.Pre vector (Baum C. et al., Mol. Ther., 13(2): 391-400 (2006); Baum C. et al., Mol. Ther., 15(6): 1167-1173 (2007)) replacing the GFP expression cassette with the NFAT promoter and hscIL12 fragment of Example 1 using the Not I and Sal I enzyme sites.

[0079]SEQ ID NO: 6 comprises the NFAT promoter located 5′ of both the IL-12 nucleotide sequence and WPRE, and the IL-12 nucleotide sequence is located 3′ of the NFAT promoter and 5′ of WPRE.

[0080]This example demonstrated a method of making a retrovirus vector comprising a nucleotide sequence encoding a nuclear factor of activated T-cells (NFAT) promoter operatively associated with a nucleotide sequence encoding human IL-12.

example 2b

[0081]The self-inactivating γ-retroviral vector pSERS11MP.GFP (Schambach et al. Mol Ther. 13:391-400 (2006); Schambach et al. Mol Ther. 15:1167-1173 (2007)) is used to construct an inducible murine single chain IL-12 vector (mflexiIL12) (SEQ ID NO: 11). To create suitable enzyme sites in pSERS11MP.GFP, primers are designed to mutate Sal I at 2771 bp and create a new Sal I site at 1550 bp (upstream of MPSV promoter) in the vector. The primers are pSERS11MP.GFPa2774t-F (SEQ ID NO: 17), pSERS11MP.GFPa2774t-R (SEQ ID NO: 18), pSERS11MP.GFP1550M-F (SEQ ID NO: 19), and pSERS11MP.GFP1550M-R (SEQ ID NO: 20). The MPSV promoter in pSERS11MP.GFP (Sal I+Nco I) is replaced by the NFAT responsive promoter from LVV-NFAT.GFP (Nco I+Xho I) to generate pSER.NFAT.GFP.

[0082]The mflexiIL12 gene is amplified by PCR from MSGV1-mflexiIL12 to introduce NcoI and Xho I restriction enzyme sites and then cut and inserted into pSER.NFAT.GFP (cut with Nco I+Sal I) to generate the construct pSER.NFAT.mflexiIL12-F....

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Abstract

The invention provides an isolated or purified nucleic acid comprising a nucleotide sequence encoding a nuclear factor of activated T-cells (NFAT) promoter operatively associated with a nucleotide sequence encoding IL-12. The invention also provides a nucleic acid comprising a nucleotide sequence encoding a nuclear factor of activated T-cells (NFAT) promoter operatively associated with a nucleotide sequence encoding IL-12, wherein the NFAT promoter is located 3′ of the nucleotide sequence encoding IL-12. Also provided are related recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. The invention further provides the use of the inventive nucleic acids or related materials in the treatment or prevention of cancer or an infectious disease in a mammal and in the induction of IL-12 expression in a mammal.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This patent application is a U.S. National Phase of International Patent Application No. PCT / US10 / 31988, filed Apr. 22, 2010, which claims the benefit of U.S. Provisional Patent Application No. 61 / 174,046, filed Apr. 30, 2009, which are incorporated by reference in their entirety herein.INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED[0002]Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 77,633 Byte ASCII (Text) file named “709090ST25.TXT,” created on Oct. 12, 2011.BACKGROUND OF THE INVENTION[0003]Interleukin 12 (IL-12) is a heterodimeric, pro-inflammatory cytokine with varied functions in the immune system. For example, IL-12 enhances cytolytic activity by inducing the production of effector cytokines, e.g., interferon-γ (IFN-γ), TNF-α, and / or granulocyte macrophage colony stimulating factor (GM-CSF). IL...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N1/15C12N1/13C12N1/21
CPCC07K14/4702C07K14/5434A61K38/00A61P31/00A61P35/00Y02A50/30
Inventor MORGAN, RICHARD A.ROSENBERG, STEVEN A.ZHANG, LINGRESTIFO, NICHOLAS P.
Owner UNITED STATES OF AMERICA
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